Polyphyly in 16S rRNA-based LVTree Versus Monophyly in Whole-genome-based CVTree.

We report an important but long-overlooked manifestation of low-resolution power of 16S rRNA sequence analysis at the species level, namely, in 16S rRNA-based phylogenetic trees polyphyletic placements of closely-related species are abundant compared to those in genome-based phylogeny. This phenomenon makes the demarcation of genera within many families ambiguous in the 16S rRNA-based taxonomy. In this study, we reconstructed phylogenetic relationship for more than ten thousand prokaryote genomes using the CVTree method, which is based on whole-genome information. And many such genera, which are polyphyletic in 16S rRNA-based trees, are well resolved as monophyletic clusters by CVTree. We believe that with genome sequencing of prokaryotes becoming a commonplace, genome-based phylogeny is doomed to play a definitive role in the construction of a natural and objective taxonomy.

On monospecific genera in prokaryotic taxonomy.

A monospecific genus contains a single species ever since it was proposed. Though formally more than half of the known prokaryotic genera are monospecific, we pick up those which actually raise taxonomic problems by violating monophyly of the taxon within which it resides. Taking monophyly as a guiding principle, our arguments are based on simultaneous support from 16S rRNA sequence analysis and whole-genome phylogeny of prokaryotes, as provided by the LVTree Viewer and CVTree Web Server, respectively. The main purpose of this study consists in calling attention to this specific way of global taxonomic analysis. Therefore, we refrain from making formal emendations for the time being.

LVTree Viewer: An Interactive Display for the All-Species Living Tree Incorporating Automatic Comparison with Prokaryotic Systematics.

We describe an interactive viewer for the All-Species Living Tree (LVTree). The viewer incorporates treeing and lineage information from the ARB-SILVA website. It allows collapsing the tree branches at different taxonomic ranks and expanding the collapsed branches as well, keeping the overall topology of the tree unchanged. It also enables the user to observe the consequence of trial lineage modifications by re-collapsing the tree. The system reports taxon statistics at all ranks automatically after each collapsing and re-collapsing. These features greatly facilitate the comparison of the 16S rRNA sequence phylogeny with prokaryotic taxonomy in a taxon by taxon manner. In view of the fact that the present prokaryotic systematics is largely based on 16S rRNA sequence analysis, the current viewer may help reveal discrepancies between phylogeny and taxonomy. As an application, we show that in the latest release of LVTree, based on 11,939 rRNA sequences, as few as 24 lineage modifications are enough to bring all but two phyla (Proteobacteria and Firmicutes) to monophyletic clusters.

CVTree3 Web Server for Whole-genome-based and Alignment-free Prokaryotic Phylogeny and Taxonomy.

A faithful phylogeny and an objective taxonomy for prokaryotes should agree with each other and ultimately follow the genome data. With the number of sequenced genomes reaching tens of thousands, both tree inference and detailed comparison with taxonomy are great challenges. We now provide one solution in the latest Release 3.0 of the alignment-free and whole-genome-based web server CVTree3. The server resides in a cluster of 64 cores and is equipped with an interactive, collapsible, and expandable tree display. It is capable of comparing the tree branching order with prokaryotic classification at all taxonomic ranks from domains down to species and strains. CVTree3 allows for inquiry by taxon names and trial on lineage modifications. In addition, it reports a summary of monophyletic and non-monophyletic taxa at all ranks as well as produces print-quality subtree figures. After giving an overview of retrospective verification of the CVTree approach, the power of the new server is described for the mega-classification of prokaryotes and determination of taxonomic placement of some newly-sequenced genomes. A few discrepancies between CVTree and 16S rRNA analyses are also summarized with regard to possible taxonomic revisions. CVTree3 is freely accessible to all users at http://tlife.fudan.edu.cn/cvtree3/ without login requirements.

Phylogeny and Taxonomy of Archaea: A Comparison of the Whole-Genome-Based CVTree Approach with 16S rRNA Sequence Analysis.

A tripartite comparison of Archaea phylogeny and taxonomy at and above the rank order is reported: (1) the whole-genome-based and alignment-free CVTree using 179 genomes; (2) the 16S rRNA analysis exemplified by the All-Species Living Tree with 366 archaeal sequences; and (3) the Second Edition of Bergey's Manual of Systematic Bacteriology complemented by some current literature. A high degree of agreement is reached at these ranks. From the newly proposed archaeal phyla, Korarchaeota, Thaumarchaeota, Nanoarchaeota and Aigarchaeota, to the recent suggestion to divide the class Halobacteria into three orders, all gain substantial support from CVTree. In addition, the CVTree helped to determine the taxonomic position of some newly sequenced genomes without proper lineage information. A few discrepancies between the CVTree and the 16S rRNA approaches call for further investigation.

On K-peptide length in composition vector phylogeny of prokaryotes.

Using an enlarged alphabet of K-tuples is the way to carry out alignment-free comparison of genomes in the composition vector (CV) approach to prokaryotic phylogeny. We summarize the known aspects concerning the choice of K and examine the results of using CVs with subtraction of a statistical background for K=3-9 and using raw CVs without subtraction for K=1-12. The criterion for evaluation consists in direct comparison with taxonomy. For prokaryotes the best performances are obtained for K=5 and 6 with subtraction and for K=11, 12 or even more without subtraction. In general, CVs with subtractions are slightly better and less CPU consuming, but CVs without subtraction may provide complementary information.

Geographic divergence of "Sulfolobus islandicus" strains assessed by genomic analyses including electronic DNA hybridization confirms they are geovars.

Ten well-annotated genomes of "Sulfolobus islandicus" strains from different geographic locations have been released at the NCBI database. Whole genome based composition vector trees indicate that these strains show the same branching patterns as originally reported by multi-locus sequence analysis. To determine whether the ten strains meet the criteria for separate species, DNA-DNA hybridization (DDH) was performed in silico. DDH values of strains from the same geographic location, i.e., Iceland, Kamchatka and North America, ranged from 82.4 to 95.4 %, clearly qualifying them as members of the same species. The lowest DDH values found between locations ranged from 75.5 to 76.6 %, which exceed the 70 % DDH threshold for a species thereby indicating they are all members of the same species based on the currently accepted definition. The clear divergences of strains from the different geographic locations are sufficiently great to consider them as separate geovars. "S. islandicus" has not yet been validly named and a type strain has not been deposited in culture collections. We urgently recommend that those who study the organism fulfill the criteria of the International Code of Nomenclature of Bacteria in order to designate a type strain and to identify and deposit related strains of this species to make them available to the broader scientific community.

Shigella strains are not clones of Escherichia coli but sister species in the genus Escherichia.

Shigella species and Escherichia coli are closely related organisms. Early phenotyping experiments and several recent molecular studies put Shigella within the species E. coli. However, the whole-genome-based, alignment-free and parameter-free CVTree approach shows convincingly that four established Shigella species, Shigella boydii, Shigella sonnei, Shigella felxneri and Shigella dysenteriae, are distinct from E. coli strains, and form sister species to E. coli within the genus Escherichia. In view of the overall success and high resolution power of the CVTree approach, this result should be taken seriously. We hope that the present report may promote further in-depth study of the Shigella-E. coli relationship.

Jackknife and bootstrap tests of the composition vector trees.

Composition vector trees (CVTrees) are inferred from whole-genome data by an alignment-free and parameter-free method. The agreement of these trees with the corresponding taxonomy provides an objective justification of the inferred phylogeny In this work, we show the stability and self-consistency of CVTrees by performing bootstrap and jackknife re-sampling tests adapted to this alignment-free approach. Our ultimate goal is to advocate the viewpoint that time-consuming statistical re-sampling tests can be avoided at all in using this alignment-free approach. Agreement with taxonomy should be taken as a major criterion to estimate prokaryotic phylogenetic trees.

Whole-genome based Archaea phylogeny and taxonomy: A composition vector approach.

The newly proposed alignment-free and parameter-free composition vector (CVtree) method has been successfully applied to infer phylogenetic relationship of viruses, chloroplasts, bacteria, and fungi from their whole-genome data. In this study we pay special attention to the phylogenetic positions of 56 Archaea genomes among which 7 species have not been listed either in Bergey's Manual of Systematic Bacteriology or in Taxonomic Outline of Bacteria and Archaea (TOBA). By inspecting the stable monophyletic branchings in CVTrees reconstructed from a total of 861 genomes (56 Archaea plus 797 Bacteria, using 8 Eukarya as outgroups) definite taxonomic assignments were proposed for these not-fully-classified species. Further development of Archaea taxonomy may verify the predicted phylogenetic results of the CVTree approach.

Composition vector approach to whole-genome-based prokaryotic phylogeny: success and foundations.

Composition vector approach to prokaryotic phylogeny provides an alignment-free and parameter-free method based on whole-genome data. It has also been applied to viruses and fungi. In all studied cases the inferred phylogenetic relationships agree well with taxonomic knowledge in major groupings and fine branchings. In this review article, after demonstrating its successful application to a collection of 892 genomes including 62 Archaea, 822 Bacteria and 8 Eukarya, we will outline some ongoing work towards the foundations of this new approach.

A fungal phylogeny based on 82 complete genomes using the composition vector method.

BACKGROUND: Molecular phylogenetics and phylogenomics have greatly revised and enriched the fungal systematics in the last two decades. Most of the analyses have been performed by comparing single or multiple orthologous gene regions. Sequence alignment has always been an essential element in tree construction. These alignment-based methods (to be called the standard methods hereafter) need independent verification in order to put the fungal Tree of Life (TOL) on a secure footing. The ever-increasing number of sequenced fungal genomes and the recent success of our newly proposed alignment-free composition vector tree (CVTree, see Methods) approach have made the verification feasible. RESULTS: In all, 82 fungal genomes covering 5 phyla were obtained from the relevant genome sequencing centers. An unscaled phylogenetic tree with 3 outgroup species was constructed by using the CVTree method. Overall, the resultant phylogeny infers all major groups in accordance with standard methods. Furthermore, the CVTree provides information on the placement of several currently unsettled groups. Within the sub-phylum Pezizomycotina, our phylogeny places the Dothideomycetes and Eurotiomycetes as sister taxa. Within the Sordariomycetes, it infers that Magnaporthe grisea and the Plectosphaerellaceae are closely related to the Sordariales and Hypocreales, respectively. Within the Eurotiales, it supports that Aspergillus nidulans is the early-branching species among the 8 aspergilli. Within the Onygenales, it groups Histoplasma and Paracoccidioides together, supporting that the Ajellomycetaceae is a distinct clade from Onygenaceae. Within the sub-phylum Saccharomycotina, the CVTree clearly resolves two clades: (1) species that translate CTG as serine instead of leucine (the CTG clade) and (2) species that have undergone whole-genome duplication (the WGD clade). It places Candida glabrata at the base of the WGD clade. CONCLUSION: Using different input data and methodology, the CVTree approach is a good complement to the standard methods. The remarkable consistency between them has brought about more confidence to the current understanding of the fungal branch of TOL.

CVTree update: a newly designed phylogenetic study platform using composition vectors and whole genomes.

The CVTree web server (http://tlife.fudan.edu.cn/cvtree) presented here is a new implementation of the whole genome-based, alignment-free composition vector (CV) method for phylogenetic analysis. It is more efficient and user-friendly than the previously published version in the 2004 web server issue of Nucleic Acids Research. The development of whole genome-based alignment-free CV method has provided an independent verification to the traditional phylogenetic analysis based on a single gene or a few genes. This new implementation attempts to meet the challenge of ever increasing amount of genome data and includes in its database more than 850 prokaryotic genomes which will be updated monthly from NCBI, and more than 80 fungal genomes collected manually from several sequencing centers. This new CVTree web server provides a faster and stable research platform. Users can upload their own sequences to find their phylogenetic position among genomes selected from the server's; inbuilt database. All sequence data used in a session may be downloaded as a compressed file. In addition to standard phylogenetic trees, users can also choose to output trees whose monophyletic branches are collapsed to various taxonomic levels. This feature is particularly useful for comparing phylogeny with taxonomy when dealing with thousands of genomes.

A simple method for phylogenomic inference using the information of gene content of genomes.

Many studies have been contributed to the inferences of phylogenies. Some studies are based on a single-gene (family), and some are based on entire genome data. In this paper, we propose a total loss genome distance approach based on gene content information to inferring phylogenies. Through various simulations, we demonstrate and evaluate the proposed approach. We compare it with some other approaches built upon gene content or extended gene content. Overall, the proposed approach performs equally well as the other methods do and is more efficient than some of the methods. We apply our approach to 34 microbial complete genomes from COG. The reconstructed tree agrees with the results from other approaches and the tree supports the concept of universal trees.

Prokaryote phylogeny meets taxonomy: an exhaustive comparison of composition vector trees with systematic bacteriology.

We perform an exhaustive, taxon by taxon, comparison of the branchings in the composition vector trees (CVTrees) inferred from 432 prokaryotic genomes available on 31 December 2006, with the bacteriologists' taxonomy--primarily the latest online Outline of the Bergey's Manual of Systematic Bacteriology. The CVTree phylogeny agrees very well with the Bergey's taxonomy in majority of fine branchings and overall structures. At the same time most of the differences between the trees and the Manual have been known to biologists to some extent and may hint at taxonomic revisions. Instead of demonstrating the overwhelming agreement this paper puts emphasis on the biological implications of the differences.

Duplication and DNA segmental loss in the rice genome: implications for diploidization.

* Large-scale duplication events have been recently uncovered in the rice genome, but different interpretations were proposed regarding the extent of the duplications. * Through analysing the 370 Mb genome sequences assembled into 12 chromosomes of Oryza sativa subspecies indica, we detected 10 duplicated blocks on all 12 chromosomes that contained 47% of the total predicted genes. Based on the phylogenetic analysis, we inferred that this was a result of a genome duplication that occurred c. 70 million years ago, supporting the polyploidy origin of the rice genome. In addition, a segmental duplication was also identified involving chromosomes 11 and 12, which occurred c. 5 million years ago. * Following the duplications, there have been large-scale chromosomal rearrangements and deletions. About 30-65% of duplicated genes were lost shortly after the duplications, leading to a rapid diploidization. * Together with other lines of evidence, we propose that polyploidization is still an ongoing process in grasses of polyploidy origins.

The tRNA and rRNA genes in the Oryza sativa genome.

In the recently assembled genomes of rice Oryza sativa ssp. indica and japonica, we identified 564 and 519 tRNA genes, respectively. The modified wobble hypothesis, namely, at least 46 tRNA species must present in order to decode all 61 possible anticodons, is perfectly observed in both subspecies. Among the 46 tRNA species, indica and japonica have many identical ones in sequence. There are 18 rice tRNA species that have identical counterparts in Arabidopsis. In the indica superscaffold dataset, 384 5S rRNA genes, dozens of 17S and 5.8S rRNA genes and one 25S rRNA gene were discovered. The incompleteness of observed rRNA genes is mainly caused by the fact that the rRNA genes always exist as tandem arrays in heterochromatic regions that are not successfully sequenced in a whole-genome shotgun approach.

Prokaryote phylogeny based on ribosomal proteins and aminoacyl tRNA synthetases by using the compositional distance approach.

In order to show that the newly developed K-string composition distance method, based on counting oligopeptide frequencies, for inferring phylogenetic relations of prokaryotes works equally well without requiring the whole proteome data, we used all ribosomal proteins and the set of aminoacyl tRNA synthetases for each species. The latter group has been known to yield inconsistent trees if used individually. Our trees are obtained without making any sequence alignment. Altogether 16 Archaea, 105 Bacteria and 2 Eucarya are represented on the tree. Most of the lower branchings agree well with the latest, 2003, Outline of the second edition of the Bergey's Manual of Systematic Bacteriology and the trees also suggest some relationships among higher taxa.

Prokaryote phylogeny without sequence alignment: from avoidance signature to composition distance.

This is a review of a new and essentially simple method of inferring phylogenetic relationships from complete genome data without using sequence alignment. The method is based on counting the appearance frequency of oligopeptides of a fixed length (up to K = 6) in the collection of protein sequences of a species. It is a method without fine adjustment and choice of genes. Applied to prokaryotic genomes it has led to results comparable with the bacteriologists' systematics as reflected in the latest 2002 outline of the Bergey's Manual of Systematic Bacteriology. The method has also been used to compare chloroplast genomes and to the phylogeny of Coronaviruses including human SARS-CoV. A key point in our approach is subtraction of a random background from the original counts by using a Markov model of order K-2 in order to highlight the shaping role of natural selection. The implications of the subtraction procedure is specially analyzed and further development of the new approach is indicated.